The central goal of this research is to characterize the proton transfer processes of two very efficient enzymes for which proton transfer is a rate-limiting step. A large part of the proposed work involves carbonic anhydrase and includes studies of the buffer-facilitated proton transfer between the enzyme active site and its environment. The utilization of this proton during the catalytic interconversion of CO2 and HCO3 will be investigated as well as related topics such as the residence time of water at the active site. The oxygen-18 exchange method allows us to determine both the rate of interconversion of CO2 and HCO3 and the rate of exchange of water from the active site at chemical equilibrium. The effect of buffer and of D2O in solvent will be used to study proton transfer in these processes. In preliminary work it has been determined that Cu2 ions at 10 to the minus 6th power M inhibits the rate of exchange of water from the active site but has no effect on the rate of interconversion of CO2 and HCO3. We will use this effect to test the hypothesis of proton transfer to the active site by measuring the kinetic properties of the copper-inhibited enzyme and by locating an approximate binding site for Cu2 ions by NMR and chemical modification of active site residues. The solvent deuterium isotope effect on the steady-state parameters will be measured as a function of deuterium content of solvent. The interpretation of these data will be refined by determining the fractionation factor for the species of water coordinated at the inner sphere of cobalt in Co(II)-carbonic anhydrase, measured by paramagnetic relaxation of water protons in H2O/D2O mixtures. The goal is to elucidate the nature of the active site, believed to be a species of water bound to the metal, and to determine the number of protons changing bounding in the transition state. These studies will be extended to membrane-bound acetylcholinesterase which exhibits a buffer effect on the rate of hydrolysis of acetylcholine very similar to that measured for the hydration of CO2 catalyzed by carbonic anhydrase. The properties of these buffer effects will be compared.